The insulin-like growth factor (“IGF”) signaling system (“IGF axis”) is comprised of the ligands IGF-I, IGF-II and insulin, and a family of transmembrane receptors including the insulin, and the type-1 and type-2 IGF receptors.
Additionally, the IGF axis includes the insulin-like growth factor binding proteins (“IGFBPs”). Six IGFBPs have been identified, cloned and sequenced. These IGFBPs share a high degree of similarity in their primary protein structure, particularly in the corresponding N- and C-terminal regions, which are separated by a variable mid-protein segment of 55 to 95 amino acid residues. The IGFBPs bind IGF-I and IGF-II, but not insulin, with high affinity. The IGFBPs appear to serve essential functions of transporting the IGFs, prolonging IGF half-lives, and regulating the availability of free IGFs for interaction with IGF receptors. Accordingly, the IGFBPs modulate the effects of IGFs on growth and differentiation. Some IGFBPs (e.g., IGFBP-3) may also be important growth-suppressing factors in various cell systems through an IGF-independent mechanism.
Other potential IGF binding proteins, referred to as IGFBP-related proteins (“IGFBP-rPs”), have been identified that have a significant similarity to the IGFBPs in their N-terminal domains. Collectively, current data supports the broad concept of an “IGFBP superfamily” with both high- and low-affinity members, wherein at least some members influence cell growth and differentiation by both IGF-dependent and IGF-independent means.
The human IGFBP superfamily is currently comprised of six high-affinity species (IGFBPs 1-6), and nine low-affinity IGFBP-related proteins (IGFBP-rPs). Structural characteristics of various members of the human IGFBP superfamily are summarized in Table 1.
TABLE 1Structural Characteristics of the Human IGFBP SuperfamilyNumberN-Chromo-Molecu-ofNumberlinkedsomalmRNAlaraminoof cys-glyco-locali-sizeIGFBPWeightacidsteinessylationzation(kb)High affinity IGFBP related proteinsIGFBP-125,27123418No7p1.6IGFBP-231,35528918No2q1.5IGFBP-328,71726418Yes7p2.4IGFBP-425,95723720Yes17q1.7IGFBP-528,55325218No2q1.7, 6.0IGFBP-622,84721616No121.1Low affinity IGFBP related proteinsIGFBP-rP1?25118Yes4q1.1IGFBP-rP2?34939Yes6q2.4(pre)IGFBP-rP3?35741? (No)8q2.4(pre)IGFBP-rP4?37935? (No)?2.4(pre)
IGFBP-3 is the principal IGFBP in adult serum, where it circulates as a 150 kDa-complex comprising IGFBP-3, an acid-labile subunit, and IGF peptide. Its principal role has been postulated to be transporting IGFs and protecting them from rapid clearance and/or degradation.
Cancer cell growth regulation. The IGFs are major regulators of mammary epithelial and breast cancer cell growth. For example, IGF-I and IGF-II are potent mitogens for a number of breast cancer cell lines in vitro. Moreover, IGF-I and IGF-II mRNAs are detectable in the majority of human breast tumor specimens. Virtually all breast tumor specimens, and cell lines derived therefrom, express and produce type-1 and type-2 IGF receptors, and insulin receptors. The mitogenic effects of both IGF-I and IGF-II are mediated by the type-1 IGF receptor, as determined through the use of estrogen-dependent breast cancer cells.
In contrast, relatively little is known about the molecular mechanisms and biological functions of the IGFBPs in the context of breast cancer. Specifically, breast cancer cells are known to secrete various types of IGFBPs, and these appear to regulate the availability of free IGFs for interaction with IGF receptors. The predominant secreted IGFBP appears to correlate with the estrogen receptor status of the cell. Estrogen-non-responsive (i.e., estrogen receptor (“ER”)-negative) cells secrete predominantly IGFBP-3 and IGFBP-4 as major species, and IGFBP-6 as a minor one. Estrogen-responsive (ER-positive) cells secrete IGFBP-2 and IGFBP-4 as major species, and IGFBP-3 and IGFBP-5 as minor ones.
Therefore, the IGF axis in breast cancer is complex, involving autocrine, paracrine, or endocrine-derived IGFs that bind to specific cellular receptors and thereby elicit, among other things, IGFBP secretion by the target cells. The IGFBPs, in turn, appear to regulate the availability of free IGFs for interaction with IGF receptors. However, the broader biological significance of IGFBPs generally, or in the particular context of breast cancer is unclear. Moreover, the basis and significance of variations in IGFBP secretory specificity are unknown, and the predominant species may vary significantly depending on the estrogen responsivess of the secreting cells.
In human breast cancer cells, expression of IGFBP-3 is hormonally regulated, and IGFBP-3 is known to inhibit cancer cell growth through (a) IGF-dependent, and (b) IGF-independent mechanisms:
(a) IGF-dependent anti-proliferative action by IGFBPs. IGFBP-3 is known to indirectly inhibit cancer cell growth through IGF-dependent interactions. IGFBP-3, as mentioned above, is the predominant IGF-binding protein in human serum where it circulates as part of a 150 kDa ternary complex. The binding affinity of IGFBP-3 for IGF peptides is generally higher than that of the type-I and type-II IGF cell-surface receptors implying that IGFBP-3 can modulate IGF binding to its receptor, thereby blocking local IGF-dependent biological action. For example, coincubation of cells with IGFBP-3 and IGF peptides results in an inhibition of the IGF-dependent mitogenic effect in human breast cancer cells, in vitro. Studies of the expression of IGF-I and IGF-II in human breast cancer tissues by in situ hybridization indicate that IGF-I mRNA is detected only in stromal cells, but not in normal or malignant epithelial cells, implying that IGF-I may function as a paracrine stimulator of epithelial cells. In contrast, IGF-II mRNA is expressed in both malignant epithelial cells and their adjacent stromal cells. Both paracrine or autocrine effects of IGF peptides can be modulated in vivo by IGFBP-3 produced by epithelial cells.
This IGF-dependent mechanism for IGFBP-3 inhibition of cancer cell growth is consistent with IGFBP-3 proteolysis studies. Post-translationally, IGFBP-3 can be proteolyzed by proteases such as cathepsin D, prostate-specific antigen (PSA) and plasmin, that are detectable in human breast cancer cells. In general, IGFBP-3 proteases are postulated to lower the affinity of IGFBP-3 for IGF, thereby increasing the availability of IGFs to cell-membrane receptors. PSA, for example, has been shown to reverse the inhibitory effect of IGFBP-3 on IGF-stimulated prostate cell growth by cleaving IGFBP-3 and generating IGFBP-3 fragments with lower affinity for IGFs. Nonetheless, the broader biological significance, molecular actions and mechanisms of IGFBP-3 proteolysis are unclear in the context of human breast cancer.
(b) IGF-independent anti-proliferative action by IGFBPs. The IGFBPs may have specific IGF-independent biological effects in various cell systems, including human breast cancer cells. For example, exogenously added IGFBP-3 inhibits estrogen-stimulated breast cancer cell proliferation. Moreover, several studies have reported that estrogen inhibits expression and secretion of IGFBP-3, whereas anti-estrogens, such as tamoxifen and ICI 182,780, stimulate production of IGFBP-3 in ER-positive human breast cancer cells.
Likewise, the expression and production of IGFBP-3 is specifically stimulated by TGF-β and retinoic acid (“RA”) in human breast cancer cells, consistent with a possible role for IGFBP-3 in TGF-β- and RA-induced growth inhibition. Additionally, the anti-proliferative effects of TGF-β, RA, TNF-α and vitamin-D analogs in human breast cancer cells appear to be partially mediated through the IGFBP-3 axis. These studies indicate that IGFBP-3 is an important IGF-independent anti-proliferative factor in human breast cancer cells.
Various experimental data support the ability of IGFBP-3 to induce apoptosis. For example, in MCF-7 cells, the treatment with recombinant human IGFBP-3 for 72 hours has been shown to increase apoptosis and to inhibit [3H]-thymidine incorporation. In Hs578T human breast cancer cells, IGFBP-3 does not result in direct induction of apoptosis, but preincubation of the cells with IGFBP-3 causes a dose-dependent potentiation of apoptosis by ceramide, an apoptosis-inducing agent, consistent with an IGF-independent activity of IGFBP-3.
Finally, IGFBP-3 can bind to the cell surface and act as a growth inhibitor for ER-negative human breast cancer cells. The interaction of IGFBP-3 with the breast cancer cell surface, and its subsequent biological effects may involve IGFBP-3-specific cell surface association proteins that mediate the direct inhibitory effect of IGFBP-3 on the growth of cells in monolayer. However, nothing is known about the nature, size, number, absolute specificity or complexity of such postulated cell surface association proteins, and no such protein has ever been cloned, purified, or otherwise characterized to provide any information whatsoever as to the actual functional involvement or sufficiency of such proteins for specific cell surface IGFBP-3 binding, or for IGFBP-3 mediated, IGF-independent biological action.
Therefore, there is a need in the art to determine the nature and mechanism of how IGFBP-3 modulates cancer cell growth and apoptosis in an IGF-independent manner. There is a further need in the art to understand how such a mechanism, once determined, could be used therapeutically, or to find therapeutic agents, or to provide useful diagnostic and prognostic assays for various cancers.